A major focus of this project is to create cellular and animals models that will allow the precise and unambiguous evaluation of the pharmacological effects of antisense and siRNA oligonucleotides and their delivery modalities. In the previous funding period the PI developed cell culture and in vivo assays in which the activity of antisense oligonucleotides results in generation of a new gene product. Antisense induced upregulation of gene expression is accomplished by targeting aberrant splice sites created in an intron inserted in the coding sequence of the EGFP gene (EGFP-654). Blocking of these splice sites with antisense oligonucleotides or RNA prevents aberrant splicing and restores correct splicing of EGFP-654 pre-mRNA, resulting in EGFP mRNA coding for a full-length, fluorescent protein. In the current project this general strategy will be modified to provide disease-oriented in vivo models for oligonucleotide actions on liver and prostate cancer. Thus we will: (a) generate liver and prostate cancer cell lines that stably express EGFP-654 gene and EGFP-WT genes;(b) generate subcutaneous xenografts of the above cells;(c) generate a cross between two transgenic mice strains, TRAMP and EGFP-654. The TRAMP mouse develops spontaneous prostate cancers, while the cross should also express the EGFP-654 pre-mRNA. This model will allow assessment of the function of antisense oligonucleotides in spontaneous tumors. In addition, (d) we will develop a new model that will allow a positive readout of siRNA and/or antisense oligonucleotides that activate RNaseH. These models, by allowing a positive read-out of antisense ands siRNA actions, provide a powerful new tool for understanding the pharmacodynamics of oligonucleotides. The models will be used to test the in vivo efficacy of various modified oligonucleotides and of oligonucleotide delivery systems.